Graphitized carbon LC-MS characterization of the chondroitin sulfate oligosaccharides of aggrecan
Graphitized carbon LC-MS characterization of the chondroitin sulfate oligosaccharides of aggrecan
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DOI：
10.1021/ac0622227
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发表时间：
2007-05-15
影响因子：
7.4
通讯作者：
Karlsson, Niclas G.
中科院分区：
文献类型：
--
作者：
Estrella, Ruby P.;Whitelock, John M.;Karlsson, Niclas G.
一种利用石墨化碳 LC-MS 研究寡糖的新型凝胶内糖苷内切酶技术揭示了聚集蛋白聚糖上硫酸软骨素的连接区和重复区之间硫酸化谱的差异。在复合琼脂糖 PAGE 凝胶中分离牛关节软骨聚集蛋白聚糖，或在醋酸铵缓冲液中稀释，并用软骨素酶 ABC 消化过夜。包括消化后的化学释放/还原方案，我们可以从三个差异硫酸化连接区六糖（Delta UA1-3GalNAc0/4/6S beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta）中分离和检测重复区域的三个差异硫酸化软骨素硫酸二糖（Delta UA1-3GalNAc0/4/6S-ol） 1-4木糖醇）。负离子模式下的石墨化碳 LC-MS 能够解析异构二糖和连接区六糖。特定的 MS2 和 MS3 使我们能够通过将所有寡糖的碎片与硫酸化二糖标准品进行比较来确认硫酸盐在所有寡糖上的位置。未硫酸化、6-硫酸化和4-硫酸化连接区的存在与消化的聚集蛋白聚糖上相应CS连接区新表位抗体（1B5、3B3、2B6）的Western blot染色阳性相关。我们检查连锁区域和重复区域概况的策略适用于从各种生物样本中筛选GAG，以检测正常状态和疾病状态之间的差异。
A novel in-gel endoglycosidase technique to study oligosaccharides with graphitized carbon LC-MS has revealed differences in the sulfation profile between the linkage and repeat regions of chondroitin sulfate on aggrecan. Bovine articular cartilage aggrecan was isolated in a composite agarose PAGE gel or diluted in ammonium acetate buffer and was digested overnight with chondroitinase ABC. Including a chemical release/reduction protocol after digestion, we could separate and detect three differentially sulfated chondroitin sulfate disaccharides of the repeat region (Delta UA1-3GalNAc0/4/6S-ol) from the three differentially sulfated linkage region hexasaccharides (Delta UA1-3GalNAc0/4/6S beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xylitol). Graphitized carbon LC-MS in the negative ion mode was able to resolve isomeric disaccharides and linkage region hexasaccharides. Specific MS2 and MS3 enabled us to confirm the sulfate location on all oligosaccharides by comparing their fragmentation with sulfated disaccharide standards. The presence of unsulfated, 6-sulfated, and 4-sulfated linkage regions was correlated with positive Western blot staining with the respective CS linkage region neoepitope antibodies (1B5, 3B3, 2B6) on digested aggrecan. Our strategy of examining linkage region and repeat region profiles is applicable to screening GAGs from various biological samples in order to detect differences between normal and disease states.